HD3a Gene inducing flowering of plant and utilization thereof

ABSTRACT

By linkage analysis, the Hd3a gene was successfully isolated. It was discovered that the flowering time of plant could be modified either by introducing the Hd3a gene or by controlling its expression.

TECHNICAL FIELD

The present invention relates to genes that induce flowering in plants,and methods for modifying the flowering time of plants using the genes.The methods for modifying the flowering time of plants are useful forplant breeding and such.

BACKGROUND ART

Generally, heading (flowering) of rice is promoted by short-dayconditions and delayed by long-day conditions. Among known cultivars,typically those from Kyushu and the south of Mainland Japan have strongphotoperiod sensitivity whereas cultivars from the Tohoku district orHokkaido show complete loss of such sensitivity or have extremely weakphotoperiod sensitivity. Rice plants that lack the photoperiodsensitivity have a characteristic to flower after a certain length ofgrowth period, and the heading date of the plant does not change withchanges in photoperiod. Adaptation of rice plants in particularlocations and seasons drastically changes in accordance with theexistence of photoperiod sensitivity in the plant. Thus, modification ofphotoperiod sensitivity in rice is an important aspect of rice breeding.

In conventional breeding programs, the alteration of the heading date ofrice is achieved through methods involving: (1) selection of earlymaturing varieties or late varieties by crossing; and (2) mutagenesis byradiation and chemicals; and so on. However, such breeding programsrequire long periods of time to be successful, and bear other problems,such as unpredictability of the degree or direction of the variation inthe progeny.

The term “photoperiod sensitivity gene” is a generic term for genes thatenhance the rice photoperiod sensitivity in the field of rice genetics.The existence of several photoperiod sensitivity genes has been observedto be inherent in mutants and cultivars, and photoperiod sensitivitygenes are suggested to exist on loci, for example, such as Sel locus(chromosome 6; Yokoo and Fujimaki (1971) Japan. J. Breed. 21:35–39), E1locus (chromosome 7; Tsai, K. H. (1976) Jpn. J. Genet. 51: 115–128;Okumoto, Y. et al. (1992) Jpn. J. Breed. 42: 415–429), E2 locus(unknown), E3 locus (chromosome 3; Okumoto et al. Japanese Society ofBreeding, 91st lecture, Japanese Journal of Breeding 47(Suppl. 1): 31),and so on (Yamagata et al. (1986) In Rice Genetics, International RiceResearch Institute, Manilla, pp351–359).

When a photoperiod sensitivity gene of rice or a gene regulating theheading of rice under the control of the photoperiod sensitivity gene isisolated, introduction of the gene into any desired rice cultivar by atransformation method makes it possible to control the heading time ofthe rice cultivar. Furthermore, the flowering time of various plants canbe controlled by utilizing genes of other plants corresponding to suchrice photoperiod sensitivity gene. This kind of breeding method can besaid to be extremely advantageous compared to conventional methods inthe aspect of convenience and reliability.

DISCLOSURE OF THE INVENTION

The present invention was made in view of these circumstances. An objectof the present invention is to provide novel genes controlling theflowering of a plant. Furthermore, another object of the presentinvention is to modify the flowering time of a plant using such genes.

The present inventors particularly focused on rice among plants, forwhich the development of a convenient method for modifying the headingtime (flowing time) is strongly desired, and actively pursued theisolation of genes associated with the heading of rice.

In rice, Hd3a, a quantitative trait loci (QTL), detected by usingprogenies derived from a cross between Nipponbare and Kasalath, had beenrevealed to be located on the short arm of chromosome 6. Furthermore,the analysis using a nearly isogenic line of Hd3a region (allele ofKasalath) having the genetic background of Nipponbare revealed that theHd3a locus is identical to a photoperiod sensitivity gene locuspromoting heading under short-day condition.

To isolate the photoperiod sensitivity gene Hd3a, which had been knownto exist but had not yet been identified, the present inventors firstmapped the Hd3a gene region by linkage analysis with P1-derivedartificial chromosome (PAC) clones. Specifically, detailed linkageanalysis of the Hd3a region was performed with a large segregatingpopulation essential for map base cloning. First, using the segregatingpopulation of the Hd3a region, a linkage map was constructed usingrestriction fragment length polymorphism (RFLP) markers and it wasproved that Hd3a was located in the interval between RELP markers C764and B174 (Monna, et al., the 1999 Annual Meeting of Japanese Society ofMolecular Biology). Furthermore, utilizing cleaved amplified polymorphicsequence (CAPS) markers CP13 and CP15 flanking both sides of Hd3a,plants having chromosomes with a recombination occurred in the vicinityof Hd3a were selected from a population having the Hd3a regionsegregated to determine their genotype by the progeny assay. Eight (8)individuals having recombination between the Hd3a and CP13 wereidentified, two (2) between Hd3a and CP15.

Next, the present inventors conducted alignment of the Hd3a gene regionusing P1-derived artificial chromosome (PAC) clones. Specifically, 3types of PAC clones having sequences of DNA markers existing in thevicinity of the Hd3a locus were screened from the Nipponbare PAC genomiclibrary. P0046E09 and P0698G05 clones had the nucleotide sequences ofmarkers, CP13 and CP15, which are flanking both sides of Hd3a, and thusthese PAC clones were revealed to comprise the Hd3a gene region (FIG.1). As a result of nucleotide sequence analysis of the PAC cloneP0046E09, the candidate genomic region was limited within a region ofabout 20 kb. Gene prediction and homology search against the nucleotidesequence of this candidate region detected regions showing high homologyto the lipid transfer protein, acyl-CoA synthase genes, and FT gene ofArabidopsis.

Then, the expression of these candidate genes was analyzed by RT-PCRusing Nipponbare and a nearly isogenic line (NIL (Hd3a)) wherein theHd3a gene region had been substituted with Kasalath chromosome segment.As a result, expression of all the three genes was confirmed, andincrease in the transcription level of the FT-like gene was observedunder short-day condition (FIG. 2). Thus, the FT-like gene was selectedas the potential Hd3a candidate gene.

A cosmid library was constructed from the genomic DNA of Kasalath. Aclone corresponding to the FT-like gene region was screened from thecosmid library (FIG. 1), and the nucleotide sequence of the FT-like generegion was analyzed. As a result, mutations at 41 sites (insertion,deletion, and substitution of nucleotides) were found in that ofKasalath as compared to the nucleotide sequence of Nipponbare (FIG. 3).A nucleotide substitution within the exon caused an amino acidsubstitution of asparagine (Kasalath) to proline (Nipponbare) (FIG. 3).

Next, the present inventors introduced a fragment containing only thecandidate gene region of Kasalath or Nipponbare into a transformablevector to transform rice plants, and analyzed the phenotype of theresulting transformant. As a result, among the plants having these genesintroduced appeared plants that showed early heading under bothshort-day and long-day conditions. However, significant changes in theheading time were not observed among plants introduced with the vectoralone (Table 1). Furthermore, self-pollinated progenies from thetransformed plants that showed early-heading under short-day conditionwere cultivated to examine the difference in the number of days fromsowing to heading (days to heading). As a result, plants showing earlyheading time compared to the control were segregated, and all the plantsshowing early heading contained the introduced gene (FIG. 4A).Similarly, under long-day conditions, in contrast to the control thatdid not reach the heading stage, heading plants was observed among theabove-described self-pollinated progenies, and all of such plantsretained the introduced fragment (FIG. 4B).

The above-described results confirmed that the candidate FT-like genehas the function to promote the heading (flowering) of rice, and it wasconcluded that the candidate FT-like gene is Hd3a gene. The Hd3a gene iswidely distributed in plants and is suggested to be associated with theinduction of flowering of these plants.

Finally, the present inventors succeeded in isolating the Hd3a gene thatinduces flowering of plant. The present inventors also found that theflowering time of plants can be modified using the gene, and completedthe present invention.

More specifically, this invention provides the following:

(1) a DNA encoding a protein derived from plants which induces theflowering of plants, wherein said DNA is selected from the groupconsisting of:

(a) a DNA encoding the protein comprising the amino acid sequence of SEQID NO: 2 or 4;

(b) a DNA encoding the protein comprising the amino acid sequence of SEQID NO: 2 or 4, wherein one or more of the amino acids are substituted,deleted, added, and/or inserted; and

(c) a DNA hybridizing under stringent conditions with the DNA consistingof the nucleotide sequence of any one of SEQ ID NO: 1, 3, 23 or 24;

(2) the DNA of (1), wherein the DNA is derived from rice;

-   -   (3) a DNA encoding an antisense RNA complementary to the        transcription product of the DNA of (1) or (2);

(4) a DNA encoding an RNA having the activity of a ribozyme thatspecifically digests the transcription product of the DNA of (1) or (2);

(5) a DNA encoding an RNA that represses the expression of the DNA of(1) or (2) upon expression in a plant cell due to a co-repressingeffect;

(6) a DNA of (1) or (2), wherein the DNA is used to induce the floweringof plant;

(7) a DNA of any one of (3) to (5), wherein the DNA is used to suppressthe flowering of plant;

(8) a vector comprising the DNA of any one of (1) to (5); (9) a plantcell introduced with the vector of (8);

(10) a plant transformant comprising the plant cell of (9);

(11) the plant transformant of (10), wherein said plant transformant isrice;

(12) a plant transformant which is a progeny or a clone of the planttransformant of (10) or (11);

(13) a breeding material of the plant transformant of any one of (10) to(12);

(14) a method for producing a plant transformant of (10) or (11), whichcomprises the following steps of:

(a) introducing the DNA of (1) or (2) into a plant cell, and

(b) regenerating a plant from the plant cell;

(15) a method for inducing the flowering of plant, wherein said methodcomprises the step of expressing the DNA of (1) or (2) in cells of theplant body;

(16) a method for suppressing the flowering of plant, which ischaracterized by the repression of the expression of endogenous DNA of(1) or (2) in cells of the plant body;

(17) the method of (16), which comprises the step of expressing the DNAof any one of (3) to (5) in cells of the plant body; and

-   -   (18) the method of any one of (14) to (17), wherein the plant is        rice.

The present invention provides DNA encoding the Hd3a protein. Thenucleotide sequences of Hd3a genomic DNA and cDNA of Kasalath are setforth in SEQ ID NO: 1 and SEQ ID NO: 23, respectively, and the aminoacid sequence of protein encoded by these DNAs in SEQ ID NO: 2.Furthermore, the nucleotide sequences of Hd3a genomic DNA and cDNA ofNipponbare are set forth in SEQ ID NO: 0.3 and SEQ ID NO: 24,respectively, and the amino acid sequence of protein encoded by theseDNAs in SEQ ID NO: 4.

Hd3a is one of quantitative trait loci (QTL) that was detected using theprogeny derived from a cross between Nipponbare and Kasalath, and wasproved to be located on the short arm of chromosome 6. Furthermore,analysis of the nearly isogenic line of the Kasalath allele of Hd3a withthe genetic background of Nipponbare revealed that the Hd3a locuspromotes heading under short-day condition.

Although Hd3a is known to be a gene having the action to promote headingunder short-day condition existing anywhere within a broad region of theshort arm of rice chromosome 6, the gene itself has not yet beenidentified or isolated. The present inventors have finally elucidatedthe existing region of Hd3a through complicated steps, succeeding forthe first time in isolating the Hd3a gene as a single gene.

Currently, the control of heading time is an important target in thebreeding of rice cultivars in Japan. In cold districts, due to the earlyarrival of autumn and low temperatures, the control of heading time iscritical to avoid cold-weather damage. On the other hand, in southwestwarm regions, to avoid concentrated harvesting labor in large scale ricegrowing zones, fine modification of heading date of rice is required.

Hd3a has the function to induce flowering. Therefore, the use of thesense strand of Hd3a gene for transformation enables the promotion ofthe heading in rice (flowering). On the other hand, the introduction ofthe gene in the antisense direction enables the suppression offlowering. The time needed for such transformation techniques in plantbreeding is remarkably short as compared to gene transfer by crossbreeding. Furthermore, the fact that the transformation does notaccompany other changes of the trait is also beneficial. Accordingly,the flowering time of a plant can be readily altered using the isolatedHd3a gene. Therefore, the gene contributes to the breeding of ricecultivars particularly adapted to different districts. Furthermore, theheading time of plants can be diversified and novel plant cultivars maybe bred for cultivars that are deficient in the Hd3a gene by introducingthe gene by genetic recombination, and for cultivars having the gene, bycontrolling the expression of the gene using antisense DNA or ribozymes.

DNAs encoding an Hd3a protein of the present invention include genomicDNAs, cDNAs, and chemically synthesized DNAs. A genomic DNA and cDNA canbe prepared according to conventional methods known to those skilled inthe art. More specifically, a genomic DNA can be prepared, for example,as follows: (1) extract genomic DNA from rice cultivars having an Hd3agene (e.g. Kasalath or Nipponbare); (2) construct a genomic library(utilizing a vector, such as plasmid, phage, cosmid, BAC, PAC, and soon); (3) spread the library; and (4) conduct colony hybridization orplaque hybridization using a probe prepared based on the DNA encoding aprotein of the present invention 30. (e.g. SEQ ID NO: 1, 3, 23, or 24).Alternatively, a genomic DNA can be prepared by PCR, using primersspecific to a DNA encoding a protein of the present invention (e.g. SEQID NO: 1, 3, 23, or 24). On the other hand, cDNA can be prepared, forexample, as follows: (1) synthesize cDNAs based on mRNAs extracted, fromrice cultivars having an Hd3a gene (e.g. Kasalath or Nipponbare); (2)prepare a cDNA library by inserting the synthesized cDNA into vectors,such as λZAP; (3) spread the cDNA library; and (4) conduct colonyhybridization or plaque hybridization as described above. Alternatively,cDNA can be also prepared by PCR.

The present invention includes DNAs encoding proteins functionallyequivalent to the Hd3a protein of SEQ ID NO: 2 or 4 (Kasalath orNipponbare). Herein, the term “functionally equivalent to the Hd3aprotein” indicates that the object protein has the function ofintroducing the flowering of plant. Such DNAs are derived preferablyfrom monocotyledonous plants, more preferably from Gramineae, and mostpreferably from rice.

Examples of such DNAs include those encoding mutants, derivatives,alleles, variants, and homologues comprising the amino acid sequence ofSEQ ID NO: 2 or 4 wherein one or more amino acids are substituted,deleted, added and/or inserted.

Examples of methods for preparing a DNA encoding a, protein comprisingaltered amino acids well known to those skilled in the art include thesite-directed mutagenesis (Kramer, W. and Fritz, H. -J., (1987)“Oligonucleotide-directed construction of mutagenesis via gapped duplexDNA.” Methods in Enzymology, 154: 350–367); The amino acid sequence of aprotein may also be mutated in nature due to the mutation of anucleotide sequence. A DNA encoding proteins having the amino acidsequence of a natural Hd3a protein wherein one or more amino acids aresubstituted, deleted, and/or added are also included in the DNA of thepresent invention, so long as they encode a protein functionallyequivalent to a natural Hd3a protein (SEQ ID NO: 2 or 4). Additionally,nucleotide sequence mutants that do not give rise to amino acid sequencechanges in the protein (degeneracy mutants) are also included in the DNAof the present invention.

Whether a DNA encodes a protein that induces flowering of a plant or notcan be assessed by, for example, as follows: cultivate the plantintroduced with a test DNA in a growth chamber wherein the photoperiodcan be modified; and examine the number of days required from sowing toflowering (from sowing to heading in rice). Under any photoperiodconditions, a protein is considered to have the floweringpromoting-function when it promotes the flowering of a plant compared toa control plant. The flowering promoting-function can be particularlyreadily proven under a photoperiod condition wherein the flowering ofthe control plant is suppressed, for example, under long-day conditions(14 to 16 h) for rice, due to the expected widened difference in theflowering time with the comparative control.

A DNA encoding a protein functionally equivalent to an Hd3a proteindescribed in SEQ ID NO: 2 or 4 can be produced, for example, by methodswell known to those skilled in the art including: hybridizationtechniques (Southern, E. M. (1975) Journal of Molecular Biology 98:503.); and polymerase chain reaction (PCR) techniques (Saiki, R. K. etal. (1985) Science 230: 1350–1354; Saiki, R. K. et al. (1988) Science239: 487–491). That is, it is routine for a person skilled in the art toisolate a DNA with high homology to the Hd3a gene from rice and otherplants using the nucleotide sequence of an Hd3a gene (SEQ ID NO: 1, 3,23, or 24) or parts thereof as a probe, and oligonucleotides hybridizingspecifically to the nucleotide sequence of the Hd3a gene (SEQ ID NO: 1,3, 23, or 24) as a primer. Such DNA encoding proteins functionallyequivalent to an Hd3a protein, obtainable by hybridization techniques orPCR techniques, are included in the DNA of this invention.

Hybridization reactions to isolate such DNAs are preferably conductedunder stringent conditions. Stringent hybridization conditions of thepresent invention include conditions such as: 6 Murea, 0.4% SDS, and0.5×SSC; and those which yield a similar stringency to the conditions.DNAs with higher homology are expected when hybridization is performedunder conditions with higher stringency, for example, 6 M urea, 0.4%SDS, and 0.1×SSC. Those DNAs isolated under such conditions are expectedto encode a protein having a high amino acid level homology with an Hd3aprotein (SEQ ID NO: 2 or 4). Herein, high homology means an identity ofat least 50% or more, more preferably 70% or more, and much morepreferably 90% or more (e.g. 95% or more), through the entire amino acidsequence.

The degree of homology of one amino acid sequence or nucleotide sequenceto another can be determined by following the algorithm BLAST by Karlinand Altschul (Proc. Nati. Acad. Sci. USA 90: 5873–5877, 1993). Programssuch as BLASTN and BLASTX developed based on this algorithm (Altschul etal. (1990) J. Mol. Biol. 215: 403–410) may be used. To analyze anucleotide sequence according to BLASTN based on BLAST, the parametersare set, for example, as score=100 and word length=12. On the otherhand, parameters used for the analysis of amino acid sequences by theBLASTX based on BLAST include, for example, score=50 and word length=3.Default parameters of each program are used when using BLAST and GappedBLAST program. Specific techniques for such analysis are known in theart.

For example, plant transformants with modified the flowering time can becreated using a DNA of the present invention. More specifically, a DNAencoding a protein of the present invention is inserted into anappropriate vector; the vector is introduced into a plant cell; andthen, the resulting transformed plant cell is regenerated. The Hd3a geneisolated by the present inventors functions to induce the flowering.Therefore, the flowering time of arbitrary cultivars can be controlledby transforming the cultivars with the gene and expressing the same. Thetime needed for transformation is remarkably short as compared toordinary gene transfer by crossing. Furthermore, the fact that thetransformation does not accompany other changes of the trait is alsobeneficial.

On the other hand, a plant transformant with repressed flowering can becreated using DNA that represses the expression of a DNA encoding aprotein of the present invention: wherein the DNA is inserted into anappropriate vector, the vector is introduced into a plant cell, andthen, the resulting transformed plant cell is regenerated. The phrase“repression of expression of DNA encoding a protein of the presentinvention” includes repression of gene transcription as well asrepression of translation to protein. It also includes not only thecomplete suppression of DNA expression but also a reduction inexpression.

The expression of a specific endogenous gene in plants can be repressedusing antisense technology methods, which are commonly used in the art.Ecker et al. were the first to demonstrate the antisense effect of anantisense RNA introduced by electroporation in plant cells using thetransient gene expression method (J. R. Ecker and R. W. Davis (1986)Proc. Natl. Acad. Sci. USA 83: 5372). Thereafter, the target geneexpression was reportedly reduced in tobacco and petunias by expressingantisense RNAs (A. R. van der Krol et al. (1988) Nature 333: 866). Theantisense technique has now been established as a means to represstarget gene expression in plants.

Multiple factors cause repression of the target gene expression byantisense nucleic acid. These include: inhibition of transcriptioninitiation resulting from triple strand formation; repression oftranscription resulting from hybrids formed at the site where the RNApolymerase has formed a local open loop structure; transcriptioninhibition resulting from hybrid formation with the RNA beingsynthesized; repression of splicing resulting from hybrid formation atthe junction between an intron and an exon; repression of splicingresulting from hybrid formation at the site of spliceosome formation;repression of mRNA translocation from the nucleus to the cytoplasmresulting from hybrid formation with mRNA; repression of splicingresulting from hybrid formation at the capping site or at the poly Aaddition site; repression of translation initiation resulting fromhybrid formation at the binding site for the translation initiationfactors; repression of translation resulting from hybrid formation atthe site for ribosome binding near the initiation codon; inhibition ofpeptide chain elongation resulting from hybrid formation in thetranslated region or at the polysome binding sites of mRNA; andrepression of gene expression resulting from hybrid formation at thesites of interaction between nucleic acids and proteins. These factorsrepress the target gene expression by inhibiting the process oftranscription, splicing, or translation (Hirashima and Inoue, “ShinSeikagaku Jikken Koza (New Biochemistry Experimentation Lectures) 2,Kakusan (Nucleic Acids) IV, Idenshi No Fukusei To Hatsugen (Replicationand Expression of Genes),” Nihon Seikagakukai Hen (The JapaneseBiochemical Society), Tokyo Kagaku Dozin, pp. 319–347, (1993)).

Accordingly, an antisense sequence of the present invention can repressthe target gene expression by any of the above mechanisms. In oneembodiment, if an antisense sequence is designed to be complementary tothe untranslated region near the 5′ end of the gene's mRNA, it willeffectively inhibit translation of a gene. It is also possible to usesequences complementary to the coding regions or to the untranslatedregion on the 3′ side. Thus, the antisense DNA used in the presentinvention includes DNA having antisense sequences against both theuntranslated regions and the translated regions of the gene. Theantisense DNA to be used is connected downstream of an appropriatepromoter, and, preferably, a sequence containing the transcriptiontermination signal is connected on the 3′ side. The DNA thus preparedcan be transfected into the desired plant by known methods. The sequenceof the antisense DNA is preferably a sequence complementary to theendogenous gene of the plant to be transformed or a part thereof, but itneed not be perfectly complementary, so long as it can effectivelyinhibit the gene expression. The transcribed RNA is preferably at least90%, and more preferably at least 95% complementary to the transcribedproducts of the target gene. In order to effectively inhibit theexpression of the target gene by means of an antisense sequence, theantisense DNA should be at least 15 nucleotides long, more preferably atleast 100 nucleotides long, and still more preferably at least 500nucleotides long. However, the antisense DNA to be used is generallyshorter than 5 kb, and preferably shorter than 2.5 kb.

DNAs encoding ribozymes can also be used to repress the expression ofendogenous genes. A ribozyme is an RNA molecule that has catalyticactivities. There are many ribozymes having various activities. Researchon ribozymes as RNA cleaving enzymes has enabled the design of aribozyme that site-specifically cleaves RNA. While some ribozymes of thegroup I intron type or the M1RNA contained in RNaseP consist of 400nucleotides or more, others belonging to the hammerhead type or thehairpin type have an activity domain of about 40 nucleotides (MakotoKoizumi and Eiko Ohtsuka (1990) Tanpakushitsu Kakusan Kohso (Nucleicacid, Protein, and Enzyme) 35: 2191).

The self-cleavage domain of a hammerhead type ribozyme cleaves at the 3′side of C15 of the sequence G13U14C15. Formation of a nucleotide pairbetween U14 and A at the ninth position is considered to be importantfor the ribozyme activity. Furthermore, it has been shown that thecleavage also occurs when the nucleotide at the 15th position is A or Uinstead of C (M. Koizumi et al. (1988) FEBS Lett. 228: 225) If thesubstrate binding site of the ribozyme is designed to be complementaryto the RNA sequences adjacent to the target site, one can create arestriction-enzyme-like RNA cleaving ribozyme which recognizes thesequence UC, UU, or UA within the target RNA (M. Koizumi et al. (1988)FEBS Lett. 239: 285; Makoto Koizumi and Eiko Ohtsuka (1990)Tanpakushitsu Kakusan Kohso (Protein, Nucleic acid, and Enzyme) 35:2191; M. Koizumi et al. (1989) Nucleic Acids Res. 17: 7059). Forexample, in the coding region of the Hd3a gene (SEQ ID NO: 1, 3, 23, or24), there are a plurality of sites that can be used as ribozymetargets.

The hairpin type ribozyme is also useful in the present invention. Ahairpin type ribozyme can be found, for example, in the minus strand ofthe satellite RNA of tobacco ringspot virus (J. M. Buzayan (1986) Nature323: 349). This ribozyme has also been shown to target-specificallycleave RNA (Y. Kikuchi and N. Sasaki (1992) Nucleic Acids Res. 19: 6751;Yo Kikuchi (1992) Kagaku To Seibutsu (Chemistry and Biology) 30: 112).

The ribozyme designed to cleave the target is fused with a promoter,such as the cauliflower mosaic virus 35S promoter, and with atranscription termination sequence, so that it will be transcribed inplant cells. However, if extra sequences have been added to the 5′ endor the 3′ end of the transcribed RNA, the ribozyme activity can be lost.In this case, one can place an additional trimming ribozyme, whichfunctions in cis to perform the trimming on the 5′ or the 3′ side of theribozyme portion, in order to precisely cut the ribozyme portion fromthe transcribed RNA containing the ribozyme (K. Taira et al. (1990)Protein Eng. 3: 733; A. M. Dzaianott and J. J. Bujarski (1989) Proc.Natl. Acad. Sci. USA 86: 4823; C. A. Grosshands and R. T. Cech (1991)Nucleic Acids Res. 19: 3875; K. Taira et al. (1991) Nucleic Acid Res.19: 5125). Multiple sites within the target gene can be cleaved byarranging these structural units in tandem to achieve greater effects(N. Yuyama et al. Biochem. Biophys. Res. Commun. 186: 1271 (1992)).Using such ribozymes, it is possible to specifically cleave thetranscription products of the target gene in the present invention,thereby repressing the expression of the gene.

Endogenous gene expression can also be repressed by co-repression,through transformation with a DNA having a sequence identical or similarto the target gene sequence. “Co-repression” refers to the phenomenonwherein, when a gene having a sequence identical or similar to thetarget endogenous gene sequence is introduced into plants bytransformation, expression of both the introduced exogenous gene and thetarget endogenous gene becomes repressed. Although the detailedmechanism of co-repression is unknown, it is frequently observed inplants (Curr. Biol. 7: R793, 1997; Curr. Biol. 6: 810, 1996). Forexample, if one wishes to obtain a plant body in which the Hd3a gene isco-repressed, the plant in question can be transformed with a vector DNAdesigned to express the Hd3a gene or DNA having a similar sequence toselect a plant with suppressed the flowering compared to wild-typeplant, among the resultant plants. The gene to be used for co-repressiondoes not need to be completely identical to the target gene, but itshould have at least 70% or more sequence identity, preferably 80% ormore sequence identity, and more preferably 90% or more (e.g. 95% ormore) sequence identity. Sequence identity may be determined by theabove-described method.

In addition, endogenous gene expression in the present invention canalso be repressed by transforming the plant with a gene having thedominant negative phenotype of the target gene. A gene having thedominant negative phenotype refers to a gene which, when expressed, caneliminate or reduce the activity of the wild type endogenous geneinherent to the plant.

Vectors used for the transformation of plant cells are not limited solong as the vector can express inserted genes in plant cells. Forexample, vectors comprising promoters for constitutive gene expressionin plant cells (e.g., cauliflower mosaic virus 35S promoter); andpromoters inducible by exogenous stimuli can be used. The term “plantcell” used herein includes various forms of plant cells, such ascultured cell suspensions, protoplasts, leaf sections, and callus.

A vector can be introduced into plant cells by known methods, such asthe polyethylene glycol method, electroporation, Agrobacterium mediatedtransfer; and particle bombardment. Plants can be regenerated fromtransformed plant cells by known methods depending on the type of theplant cell (see Toki et al., (1995) Plant Physiol. 100:1503–1507).

For example, transformation and regeneration methods for rice plantsinclude: (1) introducing genes into protoplasts using polyethyleneglycol, and regenerating the plant body (suitable for indica ricecultivars) (Datta, S. K. (1995) in “Gene Transfer To Plants”, Potrykus Iand Spangenberg Eds., pp 66–74); (2) introducing genes into protoplastsusing electric pulse, and regenerating the plant body (suitable forjaponica rice cultivars) (Tokietal. (1992) Plant Physiol. 100:1503–1507); (3) introducing genes directly into cells by the particlebombardment, and regenerating the plant body (Christou et al. (1991)Bio/Technology, 9: 957–962); (4) introducing genes using Agrobacterium,and regenerating the plant body (Hiei et al. (1994) Plant J. 6:271–282); and so on. These methods are already established in the artand are widely used in the technical field of the present invention.Such methods can be suitably used for the present invention.

Once a transformed plant, wherein a DNA of the present invention isintroduced into the genome, is obtained, it is possible to gaindescendants from that plant body by sexual or vegetative propagation.Alternatively, plants can be mass-produced from breeding materials (forexample, seeds, fruits, ears, tubers, tubercles, tubs, callus,protoplast, etc.) obtained from the plant, as well as descendants orclones thereof. Plant cells transformed with a DNA of the presentinvention, plant bodies including these cells, descendants and clones ofthe plant, as well as breeding materials obtained from the plant, itsdescendant and clones, are all included in the present invention.

The resulting plant prepared as above is different from that ofwild-type plants in terms of the flowering time. For example, plantsinto which a DNA encoding an Hd3a protein is introduced have decreasedtime to flowering under paddy field conditions. On the other hand,plants wherein the expression of a DNA encoding an Hd3a protein isrepressed due to the introduction of antisense DNAs, have delayedflowering time. Thus, the time needed for flowering of plants can beregulated by controlling the expression of the Hd3a gene. According tothe present invention, the heading date of rice, a valuable crop, can bereadily controlled, which is extremely beneficial in the breeding ofrice cultivars adapted to a particular environment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a diagram showing the high-resolution linkage map of theHd3a region and candidate genomic region thereof:

A: a linkage map prepared using segregating populations of 2207 plants;

B: Nipponbare-derived PAC clones located in the Hd3a region;

C: an enlarged map of the vicinity of the Hd3a region. Black closedcircles on the line represent CAPS markers; arrows indicate thepredicted gene regions; and Rec. shows the approximate recombinationpositions of recombinant individuals. The candidate region isrepresented by a square. DNA fragments used in the transformation arealso shown in the lower part of the map.

FIG. 2 depicts photographs showing changes in the amount of transcriptsof the FT-like gene in rice cultivated under different photoperiodconditions. “S” represents transcripts in plants that have beencultivated under long-day condition (16 h daylight) for 30 days aftersowing, then shifted to short-day treatment (10h daylight) andcultivated for 0, 2, 6, and 10 days. “L” represents transcripts fromplants that have been further cultivated for 10 days under the long-daycondition. “G” represents the genomic DNA; and “N” the controltranscript obtained without the template.

FIG. 3 depicts a diagram showing the Hd3a gene structure. Boxesrepresent the translation region. Positions of Nipponbare with differentsequence to Kasalath are shown in the figure. The transcriptioninitiation site is indicated by the arrow pointing left in the figure.

FIGS. 4A and 4B depict bar graphs showing the frequency distribution ofnumber of days to heading in the self-pollinated progenies (T1) oftransformed rice plants under the short-day (FIG. 4A) and long-day (FIG.4B) conditions, respectively. In both figures, dark bars representplants having the transgene, and bars with slanted lines those withoutthe transgene. Under the long-day conditions, no heading was observed ineither Nipponbare or NIL (Hd3a), even after 100 days.

BEST MODE FOR CARRYING OUT THE INVENTION

Herein below, the present invention is specifically described withreference to Examples; however, it is not to be construed as beinglimited thereto.

EXAMPLE 1 High-resolution Linkage Analysis

A detailed linkage analysis of the Hd3a region with a large segregatingpopulation essential for map base cloning was performed. As thesegregating population for the linkage analysis, a backcross progenyBC3F3 generation derived from a cross between Nipponbare and Kasalathwas used. The linkage analysis was carried out in two steps. First,using 595 plants of the segregating population for the Hd3a region, alinkage map was prepared with RFLP markers. As a result, Hd3a was provento be localized within the region between the RFLP markers C764 and B174(Monna, et al., The 1999 Annual Meeting, Japanese Society of MolecularBiology). Then, to further improve the map resolution, linkage analysiswas performed with a large population. Specifically, plants havingchromosomes in which recombination was occurred in the vicinity of Hd3awere screened from a population of 2207 plants segregated for the Hd3aregion using following CAPS (Cleaved Amplified Polymorphic Sequence)markers flanking Hd3a: CP13 (primer [SEQ ID NO:5/5′-GAGTAATTTGCGGTCAGAGTC-3′] and [SEQ ID NO:6/5′-CCAAACAACACATCCTCAG-3′], restriction enzyme Tai I)); and CP15(primer [SEQ ID NO: 7/5′-ACCGCAGGTCTCCTTGTCATT-3′] and [SEQ ID NO:8/5′-GCTATTGCCATCGCCTTGTGT-3′], restriction enzyme Msp I) The genotypeof the Hd3a locus was determined by the progeny testing. Specifically,10 self-pollinated progeny plants of the selected plants were cultivatedin a growth chamber under short-day conditions (irradiation for 10 h) todetermine the genotype of Hd3a base on the variation in number of daysto heading in each rice line. As a result of the linkage analysis, 8recombinant plants between Hd3a and CP13, and 2 between Hd3a and CP15were identified (FIG. 1).

EXAMPLE 2 Alignment of Hd3a Gene Region by P1-derived ArtificialChromosome (PAC) Clones

Three PAC clones (P0037G03, P0046E09, and P0698G05) having thenucleotide sequences of DNA markers 1–5A, R778, and CP39 that arelocated near the Hd3a locus were screened from the PAC genomic libraryof Nipponbare. Among the screened 3 PAC clones, P0046E09 and P0698G05were revealed to have the nucleotide sequences of Hd3a-flanking markersCP13 and CP15, and thus contain the Hd3a gene region (FIG. 1).

EXAMPLE 3 Identification of Candidate Gene Region by Nucleotide SequenceAnalysis

To further delimit the candidate genome region of Hd3a and identify thecandidate gene, the nucleotide sequence of the PAC clone P0046E09 wasanalyzed. For the nucleotide sequence analysis, the insert DNA ofP0046E09 (including the vector) was ultrasonically fragmented to preparetwo sublibraries comprising insert DNAs, one of an average length of 2kb and the other of 5 kb. Nucleotide sequences of 2000 clonesarbitrarily selected from these two types of sublibraries were analyzedand assembled using computer software Phred/Phrap. Using the informationon the nucleotide sequences within the candidate gene region specifiedby the linkage analyses, new CAPS (Cleveland Amplified PolymorphicSequence) markers were prepared to limit the candidate region. The Hd3agene co-segregated with CAPS markers 25-3UL (primers [SEQ ID NO:9/5′-TCAGAACTTCAACACCAAGG-3′] and [SEQ ID NO:10/5′-ACCTTAGCCTTGCTCAGCTA-3′], restriction enzyme Hae III) and CP39(primers [SEQ ID NO: 11/5′-GGGAGAATATGTTGCAGTAG-3′] and [SEQ ID NO:12/5′-CAAATGGTAATGGGTCAA-3′], restriction enzyme Alu I). Furthermore,two plants with recombination were detected, one with a recombinationbetween the Hd3a gene and 25-5UL (primers [SEQ ID NO:13/5′-CTGTCTCGAAATCGCCTCTG -3′] and [SEQ ID NO:14/5′-TCCAGCACATCACCCACAA-3′], restriction enzyme Hae III), and theother between the Hd3a and CP59 (primers [SEQ ID NO:15/5′-AGCCTCTGCGTCACTGTCATC-3′] and [SEQ ID NO:16/5′-GCAGCAGCAAACTCCCAAAG-3′], restriction enzyme TthH8I), respectively(FIG. 1). Thus, the candidate genomic region was delimited to a regionof approximately 20 kb. Gene prediction and similarity search wereperformed for the nucleotide sequence of this candidate region usingGENSCAN, and revealed that detect regions having a very high similarityto genes encoding the lipid transfer protein, acyl-CoA synthase, and FTgene of Arabidopsis in the candidate genomic region.

EXAMPLE 4 Expression Analysis of Hd3a Candidate Gene

RT-PCR for the candidate gene was performed for Nipponbare and thenearly isogenic line (NIL (Hd3a)) in which the Hd3a gene region issubstituted with a chromosome fragment of Kasalath. Specifically, totalRNA was extracted from collected leaves, cDNA was synthesized usingreverse transcriptase, and then PCR was performed using primer setscapable of specifically amplifying the 3 genes found within the Hd3acandidate genomic region: (for the FT-like gene: sense strand [SEQ IDNO: 9/5′-TCAGAACTTCAACACCAAGG-3′] and antisense strand [SEQ ID NO:10/5′-ACCTTAGCCTTGCTCAGCTA-3′]; for the lipid transfer protein gene:sense strand [SEQ ID NO: 17/5′-GGGGACGTCGGACCTGT-3′] and antisensestrand [SEQ ID NO: 18/5′-AGTTGAAGTTTGGGCTGGTCG-3′]; and for the acyl-CoAsynthase gene: sense strand [SEQ ID NO: 11/5′-GGGAGAATATGTTGCAGTAG-3′]and antisense strand [SEQ ID NO: 12/5′-CAAATGGTAATGGGTCAA-3′]).Assessment of the amount of RNA used as a template was carried out byPCR using a set of primers (sense strand [SEQ ID NO:19/5′-TCCATCTTGGCATCTCTCAG-3′] and antisense strand [SEQ ID NO:20/5′-GTACCCGCATCAGGCATCTG-3′]) that can amplify a fragment within theactin gene. After cultivating Nipponbare and NIL (Hd3a) for 30 daysunder long-day conditions (irradiation for 16.0 h), they were furthercultivated either under short-day condition (irradiation for 10.0 h) for0, 2, 6, and 10 days or under the long-day condition for 10 days. Then,leaves were collected from these plants for analysis.

As a result, all of the three predicted genes within the candidate generegion were confirmed to be expressed. The transcript of the FT-likegene was detected in plants cultivated under the short-day condition,but not under the long-day conditions (FIG. 2). No significantdifference in the transcript amount was observed between Nipponbare andNIL (Hd3a) under the short-day condition (FIG. 2).

As to other candidate genes, the transcript of lipid transfer proteingene was detected only in Nipponbare, and the transcript of the acyl-CoAsynthase gene was detected in both Nipponbare and NIL (Hd3a) but with nodifference in the expression level between plants cultivated under theshort-day and long-day conditions. According to the aforementionedresults, the FT-like gene whose transcription level increases under theshort-day condition was used as a potential candidate gene in functionalanalysis by transformation.

EXAMPLE 5 Nucleotide Sequence Analysis of Hd3a Candidate Gene

A cosmid library was prepared from the genomic DNA of Kasalath to screenclone H3PZ1-1 corresponding to the Hd3a candidate region. Morespecifically, using the genomic DNA of Kasalath, a genomic DNA librarywas constructed with the pPZP2CH-lac vector. The library was screenedusing the nucleotide sequences 25-5UL and CP39 that are in the vicinityof Hd3a to select the clone H3PZ1-1 containing the Hd3a candidate gene(FIG. 1). Nucleotide sequence analysis of the clone and comparisonthereof with Hd3a of Nipponbare proved the presence of nucleotidesubstitution at 32 sites, insertion (2 bp and 3 bp) at 2 sites, anddeletion (1 bp to 50 bp) at 7 sites in Nipponbare compared to theKasalath nucleotide sequence (FIG. 3).

cDNA was synthesized from RNA extracted from a plant of nearly isogenicline (NIL (Hd3a)) for cDNA nucleotide sequence analysis which plant hadbeen treated-under the short-day condition, and RT-PCR was performedusing a pair of primers comprising the region from the initiation codonto the termination codon (sense strand [SEQ ID NO:21/5′-GCTGCCTCTATCACAGTATATT-3′] and antisense strand [SEQ ID NO:10/5′-ACCTTAGCCTTGCTCAGCTA-3′]). The PCR product was cloned andsequenced. The transcription initiation site was determined by 5′-RACEto confirm that the site was 152 bp upstream of the sequence predictedas the transcription initiation codon.

As a result of the analysis, among the mutations, only a one-nucleotidesubstitution and a two-nucleotide substitution on the N-terminal sidewere found to be mutations within the exon. The latter substitution wasrevealed to cause an amino acid substitution of asparagine (Kasalath) toproline (Nipponbare) (FIG. 3).

EXAMPLE 6 Functional Identification of Candidate Gene by Transformation

8.7 kb SpeI fragment containing only the candidate gene region from thecosmid clone H3PZ1-1 was cloned into vector pPZP2H-lac that can betransformed via Agrobacterium (pPZHd3aK). Similarly, 8.7 kb SpeIfragment of Nipponbare was also incorporated into a pPZP2H-lac(pPZHd3aN). Using the vectors containing these fragments and the vectoralone, transformation was performed according to the Toki's method(Plant Mol. Biol. Rep. 15:16–21, 1997). Nipponbare was used as a riceline to be transformed. As a result, 21 (17 with the vector alone) and20 (20 with the vector alone) hygromycin-resistant plants were obtainedin the transformation experiment with pPZHd3aK and pPZHd3aN,respectively.

Whether the intended region had been incorporated was investigated byCAPS analysis with the CAPS marker 25-5UL for pPZHd3aK, and for pPZHd3aNby PCR using primer M13 Primer RV and TAKARA within the vector and theprimer within the gene [SEQ ID NO: 22/5′-CGCTCAGCAACGAGTTTC -3′]. As aresult, all of the transformants which had been introduced with pPZHd3aKand pPZHd3aN were revealed to have the candidate genes incorporated.

These regenerated plants were immediately transferred into growthchambers set up either for short-day conditions (irradiation for 10 h)or long-day conditions (irradiation for 13.5 h) to investigate thenumber of days required for heading. Among the plants introduced withpPZHd3aK and pPZHd3aN, plants showing significantly early heading wereobserved under both the short-day and long-day conditions (Table 1). Noplant with any significant changes in the heading time appeared amongthose introduced with the vector alone (Table 1).

TABLE 1 Days till heading^(a) 4 8 12 16 20 24 28 32 36 40 44 48 52 56 6064 68 72 76 80 84 88 92 96 No heading SD pPZHd3aK 1 2 4 2 Vector alone 25 3 1 LD pPZHd3aK 1 1 1 1 1 1 1 1 4 Vector alone 6 SD pPZHd3aN 1 1 2 3 3Vector alone 2 3 5 LD pPZHd3aN 1 1 2 2 1 1 2 Vector alone 1 3 1 5^(a)Days from the transplantation of transformant on soil till heading.No heading: Plants with no heading within 97 days. SD and LD:Cultivation under short-day (10.5 h daylight) and long-day (13.5 hdaylight) conditions, respectively. pPZHd3aK and pPZHd3aN: Vectoesinserted with Hd3a genes of Kasalath and Nipponbare, respectively.

Furthermore, when the self-pollinated progeny from the early-headingtransformed plant (pPZHd3aK) was cultivated under the short-daycondition to examine the segregation in the number of days to heading,the variation was in a continuous distribution, but plants showing earlyheading compared to the control Nipponbare were segregated and thesegregated plants contained the transgene (FIG. 4A).

On the other hand, when self-pollinated progenies were similarlycultivated under the long-day conditions, heading plants appeared amongthese progenies in contrast to Nipponbare and NIL (Hd3a) for which noheading is observed even after 100 days. All of the heading plantscontained the introduced DNA fragments (FIG. 4B).

These results confirmed that the FT-like gene, i.e., the candidate gene,has the function to promote the heading under the short-day conditions,and the gene was determined to be the Hd3a. The heading action of theallele of Kasalath was predicted to be stronger than that of Nipponbare.However, the allele of Nipponbare was also suggested to maintain thefunction to a degree. Early heading observed under the long-dayconditions is likely to occur due to the elevated Hd3a expression levelvia the newly introduced Hd3a gene by transformation.

INDUSTRIAL APPLICABILITY

The present invention provides genes that induce the flowering of plant.′The present invention makes it possible to control the heading date ofrice, and thus may be very useful inbreeding. Control of the headingdate of rice plants is particularly useful for breeding rice cultivarsadapted to particular locations and seasons. Furthermore, the method forbreeding rice cultivars using a gene of the present invention isbeneficial as compared to conventional methods, in that an object plantcan be obtained in a short period with high reliability.

1. An isolated DNA encoding a protein derived from plants which inducesthe flowering of plants, wherein said DNA is selected from the groupconsisting of: (a) a DNA encoding the protein comprising the amino acidsequence of SEQ ID NO: 2; and (b) a DNA encoding the protein comprisingthe amino acid sequence of SEQ ID NO: 2, wherein one amino acid issubstituted, deleted, added, and/or inserted.
 2. The DNA of claim 1,wherein the DNA is derived from rice.
 3. The DNA of claim 1, wherein theDNA is used to induce the flowering of a plant.
 4. A vector comprisingthe DNA of claim
 1. 5. A plant cell transformed with the vector of claim4.
 6. A plant transformant comprising the plant cell of claim
 5. 7. Theplant transformant of claim 6, wherein said plant transformant is rice.8. A plant transformant, which is a progeny or a clone of the planttransformant of claim 6 and comprises an isolated DNA encoding a proteinderived from plants which induces the flowering of plants, wherein saidDNA is selected from the group consisting of: (a) a DNA encoding theprotein comprising the amino acid sequence of SEQ ID NO:2; and (b) a DNAencoding the protein comprising the amino acid sequence of SEQ ID NO:2,wherein one amino acid is substituted, deleted, added, and/or inserted.9. A breeding material of the plant transformant of claim 6, whichcomprises an isolated DNA encoding a protein derived from plants whichinduces the flowering of plants, wherein said DNA is selected from thegroup consisting of: (a) a DNA encoding the protein comprising the aminoacid sequence of SEQ ID NO:2; and (b) a DNA encoding the proteincomprising the amino acid sequence of SEQ ID NO:2, wherein one aminoacid is substituted, deleted, added, and/or inserted.
 10. A method forproducing a plant transformant, which comprises the following steps of:(a) introducing the DNA of claim 1 into a plant cell, and (b)regenerating a plant from the plant cell.
 11. A method for inducing theflowering of a plant, comprising: (a) introducing the DNA of claim 1into a plant cell, and (b) regenerating a plant from the plant cell andwherein said DNA is expressed and flowering is induced.
 12. The methodof claim 10, wherein the plant is rice.